Analysis and Characterization of Cultivable Extremophilic Hydrolytic
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Journal of Applied Microbiology ISSN 1364-5072 ORIGINAL ARTICLE Analysis and characterization of cultivable extremophilic hydrolytic bacterial community in heavy-metal- contaminated soils from the Atacama Desert and their biotechnological potentials M.L. Moreno1, F. Piubeli2, M.R.L. Bonfa´ 2, M.T. Garcı´a1, L.R. Durrant2 and E. Mellado1 1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain 2 Departamento de Ciencia^ de Alimentos–FEA Universidade Estadual de Campinas-UNICAMP, Campinas, SP Brazil Keywords Abstract Atacama Desert, halophiles, halotolerants, heavy-metal-contaminated soils, hydrolytic Aims: To isolate and characterize the cultivable community of hydrolase extremophilic bacteria. producers (amylase, protease, lipase, DNase, xylanase and pullulanase) inhabiting heavy-metal-contaminated soils in extreme conditions from the Atacama Desert. Correspondence Methods and Results: A total of 25 bacterial strains showing hydrolytic Encarnacio´ n Mellado, Department of Microbi- activities have been selected including halotolerants, extremely halotolerants ology and Parasitology, Faculty of Pharmacy, and moderate halophiles. Most hydrolase producers were assigned to the University of Sevilla, C/Profesor Garcı´a Gonza´ lez, 2, 41012 Sevilla, Spain. E-mail: family Bacillaceae, belonging to the genera Bacillus (nine strains), Halobacillus [email protected] (seven strains) and Thalassobacillus (five strains) and four isolates were related to members of the families Pseudomonadaceae, Halomonadaceae and 2012/0577: received 30 March 2012, revised Staphylococcaceae. The selected strains were then characterized for their 12 June 2012 and accepted 16 June 2012 tolerance pattern to six heavy metals, measured as minimal inhibitory concentrations (MICs). doi:10.1111/j.1365-2672.2012.05366.x Conclusions: The diversity found in the cultivable bacterial community analysed is more limited than that detected in other ecological studies owing to the restrictive conditions used in the screening. The dominant bacteria were Firmicutes and particularly, species related to the genus Bacillus. Significance and Impact of the Study: This study is focused on the characterization of extremophilic hydrolytic bacteria, providing candidates as a source of novel enzymes with biotechnological applications. organic species detected in the soils (Navarro-Gonza´lez Introduction et al. 2003). In spite of the extreme conditions of this The Atacama Desert in northern Chile is a coastal desert desert habitat, a broad spectrum of halotolerant and usually described as one of the driest deserts on Earth, halophilic bacteria and archaeas have been found in salt- with a surface that has been minimally disturbed by natu- flats and hypersaline lakes in the area (Demergasso et al. ral erosion for millions of years (McKay et al. 2003; 2004; Wierzchos et al. 2006; Connon et al. 2007; De los Hartley et al. 2005). This region includes arid and semi Rı´os et al. 2010). arid environments with many different saline deposits. In Different classification schemes have been designed to these saline deposits, evaporative basins called saltflats define the relationships of micro-organisms with salt. The can be found (Chong 1984) containing a high percentage most widely accepted classification is based on the of salts from the leaching of volcanic rocks. optimal salt concentration for their growth (Kushner and Until recently, regions of the Atacama Desert were Kamekura 1988). considered the dry limit of photosynthetic activity and The halotolerance of many enzymes derived from primary production (Warren-Rhodes et al. 2006), with halotolerant and halophilic micro-organisms can be extremely low levels of cultivable organisms and oxidized exploited wherever enzymatic transformations are © 2012 The Authors 550 Journal of Applied Microbiology 113, 550--559 © 2012 The Society for Applied Microbiology M.L. Moreno et al. Extremophilic hydrolytic bacterial community required to function under extreme physical and chemical annual precipitation in AS and DV is <1 and 4 mm, conditions (Oren 2002a; Setati 2010). Hydrolases respectively, with a high UV index. At each site, we col- produced by halophiles have quite diverse potential usage lected four samples, including pieces of halite crusts bro- in different areas (food industry, feed additive, biomedi- ken off AS and surface soils (0–20 mm) from DV. cal sciences and chemical industries) (Rao et al. 1998; Samples were taken in sterile plastic containers and stored Kulkarni et al. 1999; Niehaus et al. 1999) and several in an icebox until further processed. Concentrations of halophilic enzymes have been tested for their potential Na, Ca, K, Fe, Mg, B, As, Mn, Cu and Zn were deter- biotechnological applications, including amylases, mined via ICP-OES (Inductively coupled plasma-optical nucleases and proteases (Oren 2002b). emission spectrometry) using an Iris Advantage spectrom- The Atacama Desert has increased its levels of eter (Thermo Jarrel Ash Corporation, MA, Franklin, heavy metals owing to natural and industrial processes USA). Calcium carbonate (CaCO3) was determined using (Salamanca et al. 2000). Cadmium, copper and zinc are an automated continuous flow analyser (Bran + Luebbe among those heavy metals that are being released in the III, Germany). Soil pH and electrical conductivity (EC) environment. Although some heavy metals constitute were determined in a 1 : 5 soil/water extract. An essential trace elements, most can be, at high concentra- electronic thermometer (Orion model 290) was used to tions, toxic to organisms from all branches of life, includ- measure temperature. Soil samples were air-dried, crushed ing bacteria (Nies 1999). However, some bacteria have and sieved through a 2-mm sieve. Total organic carbon adapted their physiology to tolerate the presence of metals (TOC) was analysed by dichromate oxidation and titra- or can even use them to grow. Owing to their exceptional tion with ferrous ammonium sulphate (Walkley and Black characteristics, salt tolerant bacteria adapted to live in 1934). hostile environments may exhibit the potential to remove heavy metals from contaminated environments (Mishra Isolation of hydrolytic producers et al. 2009; Zhuang et al. 2010). However, very little infor- mation is available on the heavy-metal tolerance of The isolation of micro-organisms producing hydrolytic bacteria in hypersaline environments, with the exception enzymes was performed by diluting collected samples À À of a few studies of metal-resistant halophilic bacteria from the Atacama Desert (from 10 1 to 10 3) in saline isolated from different habitats such as the Maruit Lake, solution (0Á9% NaCl, w/v) and inoculating 100 ll of the Egypt (Osman et al. 2010) or the Dead Sea Shore, Jordan dilution into saline medium (BS-10 medium) with 10% (Amoozegar et al. 2005; Massadeh et al. 2005). (w/v) total salts and 1% (w/v) yeast extract and substrates Therefore, in the present work, we studied the cultiva- to assay for amylase, protease, lipase, DNase, xylanase ble diversity of hydrolytic enzyme producers in a bacterial and pullulanase activities. The salts solution (SW) community from saline soils of the Atacama Desert. Fur- composition contained in the BS medium was as follows À1 thermore, these strains were evaluated for their biotech- (g l ): NaCl (234), MgSO4Á7H2O (61), MgCl2Á6H2O nological potentials in terms of hydrolytic enzyme (39), KCl (6), CaCl2 (1), NaBr (0Á7) and NaCO3H(0Á2). activity and heavy-metal tolerance. When necessary, different concentrations of salts solution were added to BS medium as indicated. Solid media were prepared by adding 2% (w/v) bacteriological-agar (Difco Materials and methods Laboratories, Detroit, MI). The plates were incubated at 37°C in aerobic conditions. Description of sampling sites and collection Amylase activity was determined using solid BS-10 med- Samples were collected in September 2010 from two ium supplemented with 0Á5% (w/v) soluble starch. After different points in the Atacama Desert region (Chile), des- incubation for 15 days, the plates were flooded with 0Á3% ignated as follows: Atacama Salar (AS) (in the Pre-Andean (w/v) I2 À0Á6% (v/v) KI solution: a clear zone around the Depression) (latitude 23º300′S, longitude 68º150′W) and colonies indicated hydrolysis of starch (Cowan and Steel Death Valley (DV), (latitude 22º555′S, longitude 68º120′ 1982). Protease activity was screened qualitatively in the W), both closely located to copper and lead–zinc mine saline BS-10 medium amended with 2% (w/v) skim milk. tailings. The AS is the largest saltflat in Chile and is Zones of precipitation of paracasein around the colonies located in the east region of the Antofagasta district of appearing over the next 15 days were taken as evidence of Chile. The Salar is 100 km long and 80 km wide, and the proteolytic activity (Cowan and Steel 1982). Lipase activity salts have an athalassohaline origin. The DV, also known of the strains was detected by screening for zones of hydro- as the Mars Valley, located in the Salt Mountain range, is lysis around colonies growing on solid BS-10 medium, very close to San Pedro de Atacama, standing out owing containing 1% (w/v) tributyrin after incubation for to its sand dunes of up to 150 m in height. The average 15 days (Mourey and Kilbertus 1976). The presence of © 2012 The Authors Journal of Applied Microbiology 113, 550--559 © 2012 The Society for Applied Microbiology 551 Extremophilic hydrolytic bacterial community M.L. Moreno et